The development of biopharmaceuticals as medical substances or as biotechnological products for applications in industry and science has made rapid progress during the past decades. Numerous biologically active agents selected from the classes of peptides, proteins, nucleic acids or small molecules have been identified, developed, or already been marketed.
Of major commercial interest for the development of therapeutics have been growth factors and their receptors like TNF, VEGF, or EGF. Furthermore, biologically active agents with antigen binding activity like antibodies, antibody fragments, antibody like molecules, and scaffold proteins have gained significant relevance.
The production of polyclonal antibodies is commonly known. Detailed protocols can be found for example in Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, editor), pages 1-5 (Humana Press 1992) and Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocols In Immunology, section 2.4.1 (1992). In addition, several techniques regarding the purification and concentration of polyclonal antibodies, as well as of monoclonal antibodies, are well known (Coligan et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).
The production of monoclonal antibodies is commonly known as well. Examples include the hybridoma method (Kohler and Milstein, 1975, Nature, 256:495-497, Coligan et al., section 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Despite the achievements and possibilities provided by antibodies certain disadvantages can limit the practical use. Thus, it is a problem to provide them in sufficient amounts. The production of functional antibodies is carried out in eukaryotic cell culture systems—an extraordinarily cost-intensive method. Furthermore, the low tissue penetration of the antibody molecules due to their large size and their long residence time in the serum (slow blood clearance), respectively, hamper many therapeutic applications. Although smaller fragments of antibodies such as scFv or Fab fragments (see above) can be prepared in bacteria and thus basically at lower costs, the yields of this recombinant production, however, are lower than the desired level due to their unfavourable folding properties and the required formation of several disulfide bonds. Moreover, recombinant antibody fragments often are less stable and show a lower binding activity as compared to the parental antibodies.
In order to circumvent such limitations attempts have been made to impart the principle of antibody binding—namely the binding by means of a hypervariable surface-exposed region localized on a conserved protein scaffold—to other proteins (Skerra, 2000). This means that essentially variable loops are varied in order to generate an artificial binding property. For this purpose, usually natural binding proteins such as lipocalins (Beste et al., 1999) or the fibronectin type III domain (Koide et al., 1998) have been used as a starting point for which binding sites are formed in a manner analogously to antibodies from flexible “loop” structures whose modification enables the recognition of ligands different from the natural ones.
Beside DNA-derived binding molecules, so called aptamers, a further alternative to antibodies may be binding molecules selected from the group consisting of proteins of the protein superfamily of “ubiquitin-like proteins”, in particular those having an ubiquitin-like folding motif as well as fragments or fusion proteins thereof each having the ubiquitin-like folding motif. WO 2004/106368 relates to modified proteins of this superfamily of “ubiquitin-like proteins”, proteins that have an ubiquitin-like fold. As a result of said modification, the proteins have a binding affinity with respect to a predetermined binding partner that did not exist previously. The contents of WO 2004/106368 are also incorporated herein by reference.
For scaffold derived binding molecules it is valid that the binding protein due to modifications of those amino acids forming a contiguous region on the surface of the protein, in at least one surface-exposed region of the protein preferably has a binding affinity with respect to a predetermined binding partner that did not exist previously while the original folding motif is maintained.
In summary, it turned out that a possible alternative to antibodies or aptamers thus is a group of proteins having antibody like binding behaviour.
However, there still remain major limitations for the therapeutic use of antibodies, antibody fragments, and antibody like molecules such as scaffold proteins either because of their rapid renal excretion, or poor solubility, or immunogenicity, or reduced binding affinity and/or avidity as compared with native human antibodies.
For this reason, many attempts have been made to improve the pharmacological properties of such antigen binding proteins routinely having molecular weights far below 50,000 Dalton (Da). Reviews have been published in Nature Biotechnology Volume 21, Number 4, 2006: 1126-36 or Nature Reviews Immunology, Vol 6, 2006: 343-357.
PEGylation, the covalent attachment of polyethylene glycol (PEG) to a biologically active agent, has been applied to numerous proteins and antibody fragments in order to reduce their immunogenicity and increase their circulation time in plasma (CANCER BIOTHERAPY & RADIOPHARMACEUTICALS, Volume 21, Number 4, 2006: 285-304). However, in many cases PEGylation leads to reduced target association rates via a dual blocking mechanism (Mol Pharmacol, Volume 68, 2005: 1439-1454). Chapman, A P gives a detailed overview about the divergent effects of a PEGylation for various antibodies or antibody fragments (Advanced Drug Delivery Reviews, Volume 54, 2002: 531-545).
A further approach to increase half-life and avidity of antibody like fragments has been the multimerization of two or more of such agents by introducing inter molecular disulfide bridges, peptide linkers or chemical cross-linkers. Improved tumor targeting with chemically cross-linked recombinant antibody fragments has been demonstrated for di- and trimeric Fab fragments as compared with the monomeric Fab. However, the half-life of Fab fragments could not be improved by this method (Cancer Res. (1994); 54 (23):6176-85).
Both multimerization and PEGylation represent useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting (JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 46, pp. 35186-35201, Nov. 17, 2006). However, the process of subsequently cross-linking and PEGylating an antigen binding agent is complex and thus bears certain disadvantageous with regard to yield and costs.
Consequently, attempts have been made to combine multimerization and PEGylation in one multimeric agent. In this regard, several approaches have been published.
US 2003/0211078 is related to novel pharmaceutically useful compositions that bind to a biological molecule, having improved circulatory half-life, increased avidity, increased affinity, or multifunctionality, and methods of use thereof. A pseudo-antibody is disclosed comprising an organic moiety covalently coupled to at least two target-binding moieties, wherein the target-binding moieties are selected from the group consisting of a protein, a peptide, a peptidomimetic, and a non-peptide molecule that binds to a specific targeted biological molecule. An example of such a pseudo-antibody construct shows a multimeric structure having several target binding molecules, which are linked by one single PEG moiety.
WO 03/093346 relates to high molecular weight multifunctional polyethylene glycol derivatives having a complex branched structure. From synthesis schemes 1 and 2 as well as claim 1 of WO 03/093346, it is obvious that the core structure of such a molecule results from reacting an activated PEG with a hetero-tri-functional molecule such as 2-amino-1,3-propanediol or 1,3-Diamino-2-propanol. In this way the use of such multifunctional polyethylene glycol derivatives is limited.
From US 2005/0175620 so called valency platform molecules are known comprising high molecular weight polyethylene glycol moieties, as well as conjugates thereof with biologically active molecules, and methods for their preparation. The high molecular weight polyethylene glycol moiety has, for example, a molecular weight of greater than 22,000 Daltons, for example at least 40,000 Daltons. In one embodiment, a composition comprising the valency platform molecules is provided, wherein the molecules have a polydispersity less than about 1.2. Conjugates of the valency platform molecule and a biologically active molecule, such as a saccharide, polysaccharide, amino acid, poly(amino acid), nucleic acid or lipid also are provided. Thus, this citation only describes high-molecular PEG reagents useful for prolonging half-life of comparably low molecular weight biologically active agents. However, such high-molecular PEG reagents are not suitable to increase the avidity of biologically active binding molecules, such as antibodies or antibody like proteins.
WO 2005/061005 describes branched molecular scaffolds which are capable of linking two polymer residues (derived, for example, from polyethylene glycol) to two, three or four residues derived from biologically active molecules (e.g., from whole antibodies or from functionally active fragments or derivatives thereof), the latter being attached to the scaffold by means of hydrolytically stable linkages.
WO 03049684 provides a pseudo-antibody comprising an organic moiety covalently coupled to at least two target-binding moieties, wherein the target-binding moieties are selected from the group consisting of a protein, a peptide, a peptidomimetic, and a non-peptide molecule that binds to a specific targeted biological molecule. The pseudo-antibody may affect a specific ligand in vitro, in situ and/or in vivo.
Some multimeric agents have been published using amino acids such as lysine residues as branching unit. Galande, A. K. et al. prepared multimeric imaging probes using the multiple antigenic peptide (MAP) system as the core branching unit (J. Med. Chem., Vol. 49, 2006: 4715-4720). The application of such multimeric agents is limited to special applications. Berna, M. et al. prepared monodisperse PEG-Dendrons by reacting multiple lysine residues with PEG (Biomacromolecules, Vol. 7, No. 1, 2006: 146-153). However, with increasing number of lysine residues the number of peptide bonds increases. These peptide bonds may be susceptible to hydrolysis by peptidases and furthermore to recognition by the immune system resulting in undesired side effects. Furthermore, amino acids and peptides are routinely prepared by involving microbial production processes. Thus, those basic materials bear the risk of contaminations with microbial substances such as toxins.
A multimeric agent based on PEGylated polyamidoamine (PAMAM) has been published by Yang, H. and Lopina, S. T. (J. Biomed. Mat. Res. A, Vol. 76, No. 2, 2006:398-407). PAMAM routinely bears more than 30 free amino groups and consequently is only useful for the multimerization of large numbers of biologically active molecules. Furthermore, it will hardly be possible to obtain a uniform quality with a defined number of attaching sites for a biologically active molecule based on PAMAM.
Numerous multimeric homofunctional PEG molecules have been published basing on polyalcohols as the central core unit such as glycerol or pentaerythritol. Such multimeric homofunctional PEG molecules are prepared either by Williamson ether synthesis or by ethoxylation of the hydroxyl groups of the central unit. Both synthesis strategies result in ether bonds between PEG and the central branching moiety. Related patents are cited in the following:
WO 03/033028 claims a molecule comprising a non-protein polymer, e.g. PEG, having at least three proteins linked thereto. The structure of the central non-protein polymer is based on a polyglycerol (Shearwater Polymers Inc.) as disclosed in example 1. For structural details of the non-protein polymer we refer to WO 01/62827.
WO 01/62827 of Shearwater Corporation discloses homofunctional multimeric non-peptidic polymers directly bonded to the nitrogen of an N-maleimidyl moiety. The branching unit is selected from the group consisting of glycerol, glycerol oligomers, pentaerythritol, sorbitol, and lysine. The latter is only suitable for the preparation of bi- or trivalent multimeric agents. All other branching units require the above mentioned reactions and end in an ether bond as illustrated in example 4 of WO 01/62827 for the preparation of 4-arm 10 KDa PEG maleimide.
WO 95/25763 discloses dendrimeric-type macromolecules prepared by Williamson ether synthesis with Pentaerythritol as central branching unit. Yields of such a synthesis are comparably low making this approach less attractive for commercial applications. Stein et al. published the preparation of multiple-peptide conjugates by using an eight arm branched amino-PEG from Shearwater polymers (Bioconjugate Chem. Vol 14, No. 1, 2003: 86-92). The latter is prepared by ethoxylation of a polyglycerol (for details see shearwater product catalogue, furthermore we refer to WO 01/62827)).
The before cited publications on multimeric agents based on polyalcohols all bear significant structurally determined disadvantages:                a) A Williamson ether synthesis results in essentially uniform multimeric agents, however, yield of such a reaction is very low and makes this approach unattractive for commercial applications.        b) Ethoxylation results in high yields of the final product, however, due to the polymerisation process the resulting multimeric agents routinely show unfavourable high variations in quality and furthermore are not available with defined low molecular weights.        
Despite the above-mentioned achievements, multimerization of four biologically binding molecules, in theory, can be achieved by using tetra-functionalized PEG, it is not a practical option since homogeneous tetra-functionalized PEG suitable for pharmaceutical use are not readily available (J. Immunol. Methods 2006 Vol 310 (1-2): 100-16).
In summary, there is still a great demand for multimeric agents with four or more attachment sites, which have uniform quality, variable linker length, and a defined number of reactive groups, and furthermore are capable of increasing the solubility, modulating the molecular weight, and improving the avidity of conjugates with biologically active molecules thereof.